The objective of this research program is the elucidation of the molecular mechanisms of chemical mutagenesis in mammalian cells. In the initial phases of this program, the halogenated thymidine (dT) analog 5-bromodeoxyuridine (BrdU) will be the mutagen to be studied. The cell lines to be used include a Syrian hamster melanoma line and a subline selected for viability with all the dT residues in nuclear DNA replaced by BrdU. Our results thus far suggest that the critical factor in BrdU mutagenicity is not the amount of BrdU in DNA but the concentration of BrdU to which the cells are exposed. Based on the results, our working hypothesis is that BrdU mutagenesis involves the inhibition by BrdU triphosphate of the ribonucleotide reductase catalyzed reduction of CDP to dCTP and the concomitant perturbation of deoxycytidine (dC) nucleotide metabolism. We will use a variety of genetic and biochemical techniques to try to answer the following types of questions: Do soluble BrdU nucleotides in mammalian cells induce DNA repair functions that are error-prone and therefore mutagenic? Must BrdU be present in DNA for mutagenesis to occur? Is BrdU mutagenesis mediated through the perturbation of dC nucleotide metabolism?